Human heart cells display an increase in the duration of the action potential with age. This prolongation in the action potential is possibly due to sustained elevated levels of cytoplasmic Ca2+ in cardiomyocytes. Since the Na+/Ca2+ exchanger is the major pathway of Ca2+ efflux in cardiomyocytes, we propose to study mechanisms that alter exchanger activity in aged heart. My laboratory has worked with various aspects of the exchanger: cloned the human Na+/Ca2+ exchanger (NCX1), demonstrated that alternative splicing from a single gene generates different isoforms, identified isoforms of the Na+/Ca2+ exchanger in different tissues, including heart, kidney and brain and characterized a Drosophila homologue of NCX1. Recently, we have shown that the transport activity of the Na+/Ca2+ exchanger is enhanced by phosphorylation and the effect of phosphorylation is dependent on the alternatively spliced isoforms of the exchanger. Based on these results we hypothesize that aging alters the activity of the Na+/Ca2+ exchanger either by changing the NCX1 isoforms present in heart muscle (and/) or by changing phosphorylation mechanism that can affect Na+/Ca2+ exchanger function in the heart. To test this hypothesis, we propose aims that are designed to study age-related changes in cardiomyocytes. The first aim is to characterize Ca2+ transport protein in young and aged rats by (a) measuring the amount of RNA and protein for the Na+/Ca2+ exchanger and other Ca2+ transport proteins, (b) determining the types of NCX1 isoforms and their relative proportion and (c) comparing the intracellular distribution of the Na+/Ca2+ exchanger with rats. The second aim consists of functional analysis of Na+/Ca2+ exchanger in cardiomyocytes from young and aged rats. Cardiomyocytes from young and aged rats will be compared for Na+/Ca2+ exchanger function in both the forward mode (Ca2+ efflux) and in the reverse mode (Ca2+ influx). The impact of agents that activate either the PKA or PKC phosphorylation pathways and the effect of stimulation through the beta-adrenergic receptor, which has been demonstrated to induce phosphorylation in heart cells, will be compared in young and aged rats for both modes of Na+/Ca2+ exchanger function. These results will provide important fundamental information for further study of cardiac physiology and the Na+/Ca2+ exchanger in aging.